Ubaldo Leli

Pancreatic duct obstruction in rabbits causes digestive zymogen and lysosomal enzyme colocalization.

The pancreatic duct of anesthetized rabbits was cannulated and, in some animals, flow of pancreatic exocrine secretions was blocked by raising the cannula to a vertical position. Blockage for 3-7 h caused a rapid and significant rise in serum amylase activity and an increase in amylase activity within the pancreas. The concentration of lysosomal enzymes in the pancreas was not altered but they became redistributed among subcellular fractions and, as a result, an increased amount was recovered in the 1,000-g, 15-min pellet, which was enriched in zymogen granules. Immunofluorescence studies indicated that lysosomal enzymes become localized within organelles which, in size and distribution, resemble zymogen granules. They also contain digestive enzyme zymogens. Blockage of pancreatic secretions also caused lysosomal enzyme-containing organelles to become more fragile and subject to in vitro rupture. These changes noted after short-term pancreatic duct obstruction are remarkably similar to those previously noted to occur during the early stages of diet and secretagogue-induced experimental pancreatitis, observations that have suggested that colocalization of digestive enzyme zymogens and lysosomal hydrolases might result in intracellular digestive enzyme activation and be an important early event in the evolution of those forms of experimental acute pancreatitis.

Differential Expression and Subcellular Localization of Protein Kinase C α, β, γ, δ, and ɛ Isoforms in SH-SY5Y Neuroblastoma Cells: Modifications During Differentiation

Abstract: A decrease in protein kinase C activity caused either by treatment with inhibitors, such as staurosporine or H‐7, or by prolonged exposure to phorbol diesters has been proposed to be involved in the early events of SH‐SY5Y neuroblastoma cell differentiation. Because eight distinct isoforms of protein kinase C with discrete subcellular and tissue distributions have been described, we determined which isoforms are present in SH‐SY5Y cells and studied their modifications during differentiation. The α, β, δ, and ɛ isoforms were present in SH‐SY5Y cells, as well as in rat brain. Protein kinase C‐α and ‐β1 were the most abundant isoforms in SH‐SY5Y cells, and immunoreactive protein kinase C‐δ and ‐ɛ were present in much smaller amounts than in rat brain. Subcellular fractionation and immunocytochemistry demonstrated that all four isoforms are distributed bimodally in the cytoplasm and the membranes. Immunocytochemical analysis showed that the α isoform is associated predominantly with the plasma membrane and the processes extended during treatment with 12‐tetradecanoyl‐13‐acetyl‐β‐phorbol or staurosporine, and that protein kinase C‐ɛ is predominantly membrane‐bound. Its localization did not change during differentiation. Western blots of total SH‐SY5Y cell extracts and of subcellular fractions probed with isoform‐specific polyclonal antibodies showed that when SH‐SY5Y cells acquired a morphologically differentiated phenotype, protein kinase C‐α and ‐ɛ decreased, and protein kinase C‐β1, did not change. These data suggest distinct roles for the different protein kinase C isoforms during neuronal differentiation, as well as possible involvement of protein kinase α and ɛ in neuritogenesis.

Stimulation of Phosphoinositide Hydrolysis by Serotonin in C6 Glioma Cells

Abstract: 5‐Hydroxytryptamine (serotonin or 5‐HT) stimulated the incorporation of 32Pi into phosphatidylinositol (PI) but not into polyphosphoinositides in C6 glioma cells with an EC50 of 1.2 ± 10‐7M. The phosphoinositide response was blocked by the 5‐HT2 antagonists ketanserin and spiperone but inhibited only partly by methysergide and mianserin. Atropine, prazosin, and yohimbine did not block the response, whereas fluphenazine and haloperidol did so partially but also inhibited basal incorporation by ˜30%. The 5‐HT1A agonist 8‐hydroxy‐2 (di‐n‐propylamino)tetralin did not cause stimulation. Incubation with 5‐HT (1 μM) for 1 h increased the incorporation of [2‐3H]myo‐inositol into all phosphoinositides but not into inositol phosphates (IPs). Li+ alone at 10 mM increased labeling in inositol bisphosphate (IP2) and trisphosphate (IP3), whereas labeling in IP and phosphoinositides remained unaltered. Addition of 5‐HT had no effect on this increase. Mn2+ at 1 mM enhanced labeling in PI, PI‐4‐phosphate, lyso‐PI, glycerophosphoinositol, and IP, but the presence of 5‐HT again did not cause further stimulation. 5‐HT also stimulated the release of IPs in cells prelabeled with [2‐3H]myo‐inositol, incubated with LiCl (10 mM) and inositol (10 mM), and then exposed to 5‐HT (1 μM). Radioactivity in IP2 and IP3 was very low, was stimulated ˜50% as early as 30 s, and remained elevated for at least 20 min. Radioactivity in IP was at least 10 times as high as in IP3 but was increased only from 3 min on with a peak at 20 min, when the elevation was ˜40 times that in IP3. The EC50 value of 1.8 ± 10‐7M was comparable with that obtained from 32Pi studies measuring labeled PI. Ketanserin and spiperone inhibited 5‐HT‐stimulated [2‐3H]IP release with IC50 values of 3.1 ± 10‐9 and 1.8 ± 10, ‐8M, respectively. This study demonstrates that phosphoinositide hydrolysis is enhanced by 5‐HT in C6 glioma cells and that this phenomenon is linked to 5‐HT2‐like binding sites.

Intracellular delivery of protein kinase C-α or ε isoform-specific antibodies promotes acquisition of a morphologically differentiated phenotype in neuroblastoma cells

The protein kinase C (PKC) family participates in a ubiquitous cell signalling system utilizing increased turnover of phosphoinositides. Because down‐regulation of total PKC activity has been implicated in the acquisition of a morphologically differentiated phenotype in SH‐SY5Y neuroblastoma cells, we aimed to identify the specific PKC isoforms in this process. Here we report that intracellular delivery of PKC‐α and ‐ε, but not ‐β|4‐γ or ‐δ isoform‐specific antibodies is sufficient to induce acquisition of a morphologically differentiated phenotype in SH‐SY5Y neuroblastoma cells.

Esterase inhibitors prevent lysosomal enzyme redistribution in two noninvasive models of experimental pancreatitis

Earlier studies have indicated that lysosomal enzymes such as cathepsin B become redistributed within pancreatic acinar cells during the early stages of both diet- and secretagogue-induced acute pancreatitis. As a result, cathepsin B and digestive zymogens became colocalized within large cytoplasmic vacuoles. As cathepsin B can activate trypsinogen, this colocalization could result in intracellular digestive enzyme activation. The present study investigates the protective effects of gabexate mesilate (FOY) and camostate (FOY 305) on both of these noninvasive models of experimental pancreatitis. These esterase inhibitors prevented the hyperamylasemia, pancreatic edema, and acinar cell vacuolization that characterize secretagogue-induced pancreatitis and the hyperamylasemia and mortality that characterize diet-induced pancreatitis. In addition, FOY and FOY 305 were found to significantly decrease the subcellular redistribution of cathepsin B that occurs in both models. These findings indicate that enzyme activity sensitive to inhibition by FOY and FOY 305 may be critical to the redistribution phenomenon that characterizes both of these models of pancreatitis.

Modifications of phospholipid metabolism induced by chlorpromazine, desmethylimipramine and propranolol in C6 glioma cells

The effects of chlorpromazine (CPZ), desmethylimipramine (DMI) and propranolol (PRO) on phospholipid metabolism in C6 glioma cells were studied by following the incorporation of 32Pi, [U-14C]glycerol, [2-3H]glycerol and [1-14C]oleate into lipids. The drugs produced a dose-dependent increase in the incorporation of 32Pi and [U-14C]glycerol, but not of [1-14C] oleate, into total phospholipids, that reached a plateau at 200 microM CPZ and 500 microM DMI and PRO. The three drugs shifted the incorporation of precursors from neutral [phosphatidylcholine (PC) and phosphatidylethanolamine (PE)] to acidic phospholipids [phosphatidic acid (PA), phosphatidylinositol (PI), phosphatidylglycerol, phosphatidylinositol-4-phosphate (PIP) and phosphatidylinositol-4,5-bisphosphate (PIP2)] in a dose-dependent, qualitatively similar manner. The incorporation of [2-3H]glycerol into diacylglycerol was also depressed markedly by CPZ. Addition of 1 mM 1,2-dioleoylglycerol, 1-oleoyl-2-acetylglycerol or oleate only partially reversed the decrease in PC labeling caused by CPZ. 12-O-Tetradecanoylphorbol-13-acetate counteracted this effect of CPZ completely but greatly increased PC labeling even in the absence of the drug. Polyphosphoinositides rapidly incorporated 32Pi at early times reaching a plateau in about 40 min. The labeling rate of PI was not parallel to that of PIP or PIP2 and continued to increase even after the polyphosphoinositides had reached a plateau. CPZ increased PI labeling much more than that of PIP and PIP2. These data suggest that cationic amphiphilic drugs may act by inhibiting CTP:phosphocholine cytidylyltransferase, thus decreasing incorporation of precursors into PC and PE; inhibiting PA phosphohydrolase with increased formation of phosphatidyl-CMP, the intermediate for the synthesis of acidic phospholipids; and stimulating the inositol exchange reaction, forming a pool of PI that is not available for PIP and PIP2 synthesis.

Influence of lithium on second messenger accumulation in NG 108-15 cells

Treatment of NG108-15 cells with bradykinin for 10 min resulted in a reduction of phosphatidylinositol levels with no change in the other inositol lipids. Accompanying this was a small increase in diacylglycerol but no modification in the level of inositol phosphates. Addition of bradykinin to cells incubated for 30 min in buffer containing lithium did not cause a further decrease in the labeling of phosphatidylinositol, but was accompanied by an increase in mass of the three inositol phosphates. The increase was about 150% for inositol monophosphate, 40% for inositol bisphosphate and 80% for inositol trisphosphate. Under these conditions there was a marked increase in the ability of bradykinin to elevate the content of diacylglycerol. Moreover lithium by itself induced an increase in diacylglycerol six times as great as the increase in total inositol phosphates. The excess diacylglycerol may have its origin in phospholipids other than phosphoinositides.

Failure of a potent cholecystokinin antagonist to protect against diet-induced pancreatitis in mice.

The effects of a potent cholecystokinin (CCK) receptor antagonist, L-364,718, on two forms of experimental acute pancreatitis in mice were evaluated. The antagonist prevented the hyperamylasemia, pancreatic edema, and acinar cell vacuolization that followed administration of a supramaximally stimulating dose of the cholecystokinin analogue cerulein. In contrast, the same dose of L-364,718 (1 mg/kg/6h) and an even higher dose (10 mglkg/6 h) failed to prevent the hyperamylasemia, acinar cell necrosis, and mortality that followed administration of a choline-deficient ethionine-supplemented diet. These observations are at variance with those previously reported to follow administration of the relatively weak cholecystokinin antagonist proglumide (Niederau C et al. J Clin Znvest 1986;78: 10.5643). The observations reported in this communication suggest that cholecystokinin does not play an important role in diet-induced pancreatitis and that CCK receptor antagonists are unlikely to be of benefit in the treatment of clinical acute pancreatitis.

Chlorpromazine induces accumulation of inositol phosphates in C6 glioma cells

The capacity to modify the incorporation of [2-3H]myo-inositol into inositides and inositol phosphates was different for three psychotropic cationic amphiphilic drugs. Chlorpromazine, desmethylimipramine and propranolol were able to increase the labeling of inositol-containing lipids, but only chlorpromazine dramatically increased the incorporation into inositol phosphate, -bisphosphate and -trisphosphate. The increase was 10- to 50-fold in 60 min as compared with controls. This effect is not due to stimulation of lipid labeling, because in chase experiments radioactivity in inositol phosphates increased to a greater extent than in their parent lipids. It is possible that the alteration of phosphoinositide catabolism is related to the neuroleptic activity of the drug.

Effects of hemorrhagic shock, aspirin, and ethanol on secretagogue-induced experimental pancreatitis

SummaryThe effects of hemorrhagic shock, aspirin, and ethanol on the biochemical and morphologic changes of experimental pancreatitis were evaluated. Pancreatitis was induced by infusing rats with a supramaximally stimulating dose (5 µg/kg/h) of caerulein. Hemorrhagic shock was established by removing sufficient blood to reduce mean arterial pressure by 30%, where it was maintained for 30 min. Aspirin (25 mg/kg) and ethanol (2 g/kg) were administered through an orogastric tube at 8-h intervals for 48 h.Hemorrhagic shock did not alter the degree of hyperamylasemia, pancreatic edema, cathepsin subcellular redistribution, or in vitro LDH leakage that characterize this model of pancreatitis. Hemorrhagic shock did, however, worsen the morphologic evidence of pancreatic injury. Administration of aspirin with ethanol did not alter the degree of hyperamy-lasemia, pancreatic edema, or subcellular cathepsin redistribution. Aspirin-ethanol pretreatment also did not alter the morphologic severity of pancreatitis.These observations indicate that hemorrhagic shock worsens the microscopic evidence of pancreatitis induced by supramaximal secretagogue stimulation. In contrast, aspirin-ethanol pretreatment, which might have been expected to increase pancreatic ductal permeability, did not alter the severity of this model of experimental pancreatitis.